JP2000505285A - Human DNase - Google Patents

Abstract

This invention relates to a novel human deoxyribonuclease, referred to as LS-DNase, that is relatively resistant to inhibition by actin, as compared to human DNase I. The invention provides nucleic acid sequences encoding LS-DNase, thereby enabling the production of LS-DNase by recombinant DNA methods in quantities sufficient for clinical use. The invention also relates to pharmaceutical compositions and therapeutic uses of LS-DNase.

Description

The present invention relates to a newly identified human deoxyribonuclease (DNase), a nucleic acid encoding the protein, the use of the protein and the nucleic acid, as well as the protein, eg, a recombinant DNA method. And nucleic acid production. BACKGROUND OF THE INVENTION Deoxyribonuclease (DNase) is a phosphodiesterase capable of hydrolyzing polydeoxyribonucleic acid and is known to exist in several molecular forms. Based on their biochemical properties and enzymatic activity, DNase proteins have been classified into two types, DNase I and DNase II. The DNase I protein has an essential pH near neutral, an essential need for divalent cations, and produces 5'-phosphate nucleotides by hydrolysis of DNA. DNase II proteins exhibit an acidic pH optimum, can be activated by divalent cations, and produce 3'-phosphate nucleotides by hydrolysis of DNA. DNases from various species have been purified to varying degrees. For example, various forms of bovine DNase I have been generated and fully sequenced (Liao et al., J. Am. Biol. Che m. 248: 1489-1495 (1973); Oefner et al., J. Hol. Biol. 192: 605-632 (1986); Lahm et al., J. Mol. BiOol. 221: 645-667 (1991)), and DNA encoding bovine DNase I has been cloned and expressed (Worrall et al., J. Am. Biol. Chem 265: 21889-21895 (1990)). Porcine and orcine DNase I proteins have also been isolated and fully sequenced (Paudel et al., J. Am. Biol. Chem. 261: 16006-16011 (1986); Paudel et al., J. Biol. Chem. 261: 160 12-16017 (1986)). DNA encoding human DNase I has been isolated and sequenced, and the DNA is expressed in recombinant host cells, thereby allowing the production of human DNase I with commercially useful quality. Shak et al., Proc. Natl. Acad. Sci. 87: 9188-9192 (1990). The term "human DNase I" is used below to refer to the mature polypeptide disclosed in Shak et al. DNAs encoding other polypeptides having homology to human DNase I have also been identified. Rosen et al., PCT Patent Application No. WO 95/30428; Parrish et al., Hum. Mol. Genet. 4: 1557-1564 (1995). DNase I has many well-known utilities and has been used for therapeutic purposes. Its primary therapeutic use is to reduce the viscoelasticity of pulmonary secretions (mucus) in diseases such as pneumonia and cystic fibrosis (CF), thereby helping to clean the respiratory tract. is there. For example, Lourenco et al., Arch. Intern. Med. 142: 2299-2308 (1982); Shak et al., ProcNatl. Acad. Sci. 87: 9188-9192 (1990); Hubbard et al., New Engl. J. Med. 326: 81 2-815 (1992); Fuchs et al., New Engl. J. Hed. 331: 637-642 (1994); Bryson et al., Drugs 48: 894-906 (1994). Mucus also causes chronic bronchitis, asthmatic bronchitis, bronchiectasis, emphysema, acute and chronic sinusitis, and common colds, and contributes to the morbidity of each of these diseases. The lung secretions of humans with the disease are complex substances, including mucus glycoproteins, mucopolysaccharides, proteases, actin, and DNA. DNase I is effective in reducing the viscoelasticity of pulmonary secretions by hydrolyzing or degrading high molecular weight DNA present in the secretions. Shak et al., Proc. Natl. Acad. Sci. 87: 9188-9192 (1990); Aitken et al., J. Am. Med. Assoc. 267: 1947-1951 (1992). However, the DNA hydrolytic activity of DNase I in pulmonary secretions will be reduced as a result of the interaction of DNase I with actin. Lazarides et al., Proc. Natl. Acad. Sci. 71: 4742-4766 (1974); Hannherz et al., Eur. J. Biochem. 104: 367-379 (1980). Thus, although it binds actin with lower affinity than human native DNase I, its DNA hydrolyzing activity is a form of DNase I that is now possessed by therapeutic agents, especially pulmonary secretions containing relatively high amounts of actin. Should be useful in treating patients with Various human DNase I with reduced affinity for actin have been prepared synthetically and have been shown to be more potent than the native enzyme in reducing the viscosity of sputum in cystic fibrosis patients. ing. Lazarus et al., PCT application WO 96/26279, printed Aug. 29, 1996. SUMMARY OF THE INVENTION The present invention provides novel DNases and, at the same time, analogs and variants of DNases that have DNA hydrolytic activity but are resistant to inhibition by actin. This novel polypeptide, also called LS-DNase, is of human origin. The present invention also provides a nucleic acid encoding an LS-DNase, a recombinant vector containing the nucleic acid, a recombinant host cell transfected with the nucleic acid or the vector, and the production of LS-DNase by a recombinant DNA method. To provide a process for The invention includes the use of the nucleic acids and vectors for in vivo or ex vivo gene therapy. The invention also includes a pharmaceutical composition comprising an LS-DNase, optionally a pharmaceutically acceptable excipient, as well as a substantially purified antibody capable of binding to the LS-DNase. . The present invention also provides a method for reducing the viscoelasticity or viscosity of a DNA-containing substance in a patient, comprising administering to the patient a therapeutically effective amount of LS-DNase. The present invention particularly includes administering to a patient a therapeutically effective amount of LS-DNase, the treatment of cystic fibrosis, chronic bronchitis, pneumonia, bronchiectasis, emphysema, asthma, or systemic lupus erythematosus. And a method of treating a patient having such a disease. The present invention is also directed to the use of LS-DNase in an in vitro diagnostic assay for mucus (eg, sputum) from a patient. These and other aspects of the present invention will be apparent to one of ordinary skill in the art in view of the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGSFIG. 1 shows the nucleic acid sequence of LS-DNase (SEQ. ID. NO: 1) and the applicable amino acid sequence (SEQ. ID. NO: 2). The predicted leader (signal) amino acid sequence is underlined and the start site of the mature protein is indicated by the arrow. FIG. 2 shows human LS-DNase (SEQ. ID. NO: 3) and human DNase I (SEQ. ID. The comparison of the amino acid sequence of NO: 4) is shown. Identical amino acid residues are boxed, conservative amino acid substitutions are indicated by dots (•), and conservative cysteine residues are indicated by arrows. The two potential glycosylation sites of human DNase I involved in actin binding are shaded. Conservative catalyst residues are painted black. FIG. 3 shows the nucleic acid sequence of murine LS-DNase (SEQ. ID. NO: 11). The ATG start codon of the predicted protein is indicated by an arrow, and the nucleic acid sequence encoding the predicted leader (signal) amino acid sequence of the protein is underlined. DETAILED DESCRIPTION Various aspects of the invention are accomplished by first providing an isolated DNA comprising a nucleic acid coding sequence for an LS-DNase. By providing the full nucleic acid coding sequence for LS-DNase, the present invention enables the production of LS-DNase using recombinant DNA techniques, thereby substantially purifying it for diagnostic and therapeutic uses. Only the sufficient quality of the LS-DNase protein obtained is available. As used herein, the term "LS-DNase" refers to a polypeptide having the amino acid sequence of the mature protein shown in FIG. 1, as well as modifications and variants thereof as described herein. The term "human LS-DNase" refers to a polypeptide having the amino acid sequence of the mature protein shown in FIG. Modifications and variants of LS-DAase are produced by chemical or enzymatic treatments in vitro or by recombinant DNA methods in vivo. The polypeptide may differ from human LS-DNase, for example, by the substitution, insertion and / or deletion of one or more amino acids, or in the extent or pattern of glycosylation, The activity is substantially maintained. Preferably, the modifications and variants of LS-DNase have a DNA hydrolysis activity substantially similar to that of human LS-DNase. The term "variant" or "amino acid sequence variant" of LS-DNase is a polypeptide comprising an amino acid sequence that differs from that of human LS-DNase. Generally, a variant has at least 80% sequence identity (homology) with LS-DNase, preferably at least 90% sequence identity, more preferably 95% sequence identity, and most preferably 98% sequence identity. Will have identity. % Sequence identity, after aligning the sequence providing the greatest homology, for example, Fitch et al., Proc. Natl. Acad. Sci. USA 80: 1382-1386 (1983), or Needleman et al., J. Mol. Biol. 48: 443-453 (1970). The variants include naturally occurring allelic forms of human LS-DNase of human origin, as well as naturally occurring homologs of human LS-DNase found in other animal species. The term "DNA hydrolytic activity" refers to the enzymatic activity of a DNase to hydrolyze (cleave) substrate DNA to produce a 5'-phosphorylated oligonucleotide terminal product. DNA hydrolysis activity is readily measured using any of several different methods known in the art, including analytical polyacrylamide and agarose gel electrophoresis, dark color assays (Kurnick, Arch. Biochem. 29: 41-53 (1950); Sinicropi et al., Anal. Biochem. 222: 351-358 (1994)). For simplicity, substitutions, insertions, and / or deletions in the amino acid sequence of human LS-DNase may result in mutations in the corresponding nucleic acid sequence of the DNA encoding human LS-DNase, for example, site-directed mutagenesis. It is usually created by introducing. Expression of the mutated DNA then causes the production of a mutant LS-DNase having the desired amino acid sequence. For performing site-directed mutagenesis in any known manner, for example, as disclosed in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, New York (1989)). Whilst it can be used, oligonucleotide directed mutagenesis is a preferred method for preparing the LS-DNase variants of the present invention. Well known in the art (Zoller et al., Meth. Enzymol. 100: 4668-500 (1983); Zoller et al., Heth. Enzy mol. 154: 329-350 (1987); Carter, Meth. Enzymol. 154: 382-403 (1987); Kunkel et al., Net h. Enzymol. 154: 367-382 (1987); Horwitz et al., Meth. Enzymol. 185: 599-611 (1990)) Although this method is particularly suitable for making substitution variants, it can also be used to conveniently prepare deletion and insertion variants, as well as multiple substitution variants. Mutants with mutated, inserted, and / or deleted mutations may also be prepared. Briefly, when performing site-directed mutagenesis of DNA encoding human LS-DNase (or a variant thereof), the DNA is ligated with an oligonucleotide encoding a desired mutation to a single-stranded DNA. Modify by first hybridizing. After hybridization, a DNA polymerase is used to synthesize a complete second strand using the hybridized oligonucleotide as a primer and a single strand of the DNA as a template. Thus, the oligonucleotide encoding the desired mutation is incorporated into the resulting double-stranded DNA. Oligonucleotides are prepared by any suitable method, such as by purification of naturally occurring DNA or by in vitro synthesis. For example, oligonucleotides are described by Narang et al., Meth. Enzymol. 68: 90-98; Brown et al., Meth. Enzymol. 68: 109-151 (1979); Car uthers et al., Meth. Enzymol. 154: 287-313 (1985), and are readily synthesized using various methods in organic chemistry. General approaches for selecting suitable oligonucleotides for use in site-directed mutagenesis are well known. Typically, the oligonucleotide comprises 10-25 or more nucleic acids, to ensure that the oligonucleotide preferably hybridizes at the desired location relative to the single-stranded DNA template molecule. It will contain at least 5 nucleic acids on each side of the sequence encoding the desired mutation. The terms "polymerase chain reaction" or "PCR" generally refer to a method for the amplification of a desired nucleic acid sequence in vitro, for example, as described in US Pat. No. 4,683,195. Generally, the PCR method involves repeated cycles of primer extension synthesis using oligonucleotide primers preferably hybridizable to a template nucleic acid. PCR Mutagenesis (Higuchi, in PCR Protocols, pp. 177-183 (Academic Press, 1990); Vallette et al., Nuc. Acids Res. 17: 723-733 (1989)) are also suitable for generating variants of LS-DNase. Briefly, when a small amount of template DNA is used as a starting material in a PCR, primers that differ slightly in sequence from the corresponding region in the template DNA can be used to produce relatively large amounts of a specific DNA fragment, The specific DNA fragment differs from the template sequence only at positions where the primer differs from the template. For introduction of the mutation into the plasmid DNA, for example, one primer sequence may contain the desired mutation and be designed to hybridize to one strand of the plasmid DNA at the mutation position; The sequence must be equal to the nucleic acid sequence in the opposite strand of the plasmid DNA, but this sequence can be located anywhere on the plasmid DNA. However, the sequence of the second primer is located within 200 nucleic acids of the sequence of the first primer so that the complete amplified region of DNA ultimately bound by the primer can be easily sequenced. PCR amplification using primer pairs such as those described here results in a population of different DNA fragments at the mutation locations specified by the primers and at other positions due to some error in the template copy. . Wagner et al., In PCR Topics, pp. 69-71 (Springer-Verlag, 1991). If the ratio of template to amplified DNA product is extremely low, most of the product DNA fragments will incorporate the desired mutation (s). This product DNA is used to replace the corresponding region in the plasmid used as a PCR template using standard recombinant DNA techniques. Mutations at separate positions can be performed using the second primer of the mutant, or by performing a second PCR using a different mutant primer and simultaneously ligation of the 3 (or more) portions to the plasmid fragment. Either of the results of step 2 and the resulting PCR fragment can be ligated, either simultaneously. Another method for preparing variants, cassette mutagenesis, is based on the method described in Wells et al., Gene, 34: 315-323 (1985). The starting material is a plasmid (or other vector) containing the mutated DNA sequence. There must be a unique restriction endonuclease site on each side of the mutation site (s) identified. If the restriction sites are not present, they can be produced using the oligonucleotide-mediated mutagenesis method described above to introduce them at the appropriate location in the DNA. Cleavage at these sites to linearize the plasmid DNA. A double-stranded oligonucleotide encoding the sequence of the DNA between the restriction sites, but containing the desired mutation (s), is synthesized using standard methods, where the duplexes of the oligonucleotide are separated. And hybridized together using standard methods. This double-stranded oligonucleotide is called a cassette. This cassette is designed to have 5 'and 3' ends that match the ends of the linearized plasmid so that it can be directly ligated to the plasmid. The resulting plasmid contains the mutated DNA sequence. The presence of mutation (s) in DNA is determined by methods well known in the art, including restriction mapping and / or DNA sequencing. Preferred methods for DNA sequencing are described in Sanger et al., Proc. Natl. Acad. Sci. USA 72: 3918-3921 (1979). DNA encoding LS-DNase is inserted into a replicating vector for further cloning or expression. The term "vector" refers to plasmids and other DNA that are capable of replication in a host cell and are also useful for performing compatible functions with compatible host cells (vector-host systems). is there. One function is to facilitate cloning of the nucleic acid encoding the LS-DNase, ie, to produce usable quantities of the nucleic acid. Another function is to direct the expression of LS-DNase. One or two of these functions are performed by the vector in the particular host cell used for cloning or expression. The vectors will contain different components depending on the function they perform. The LS-DNase of the present invention may be in the form of a pre-protein containing a leader or signal sequence, or in the form of a mature protein lacking a leader or signal sequence. The LS-DNase may also be in the form of a fusion protein in which additional amino acid residues are covalently linked to the amino or carboxy terminus of the DNase preprotein or mature form. To produce LS-DNase, the expression vector will include a DNA encoding the LS-DNase operably linked to a promoter and a ribosome binding site, as described above. The LS-DNase is then expressed directly in recombinant cell culture, or preferably, having a specific cleavage site at the junction between the signal sequence or heterologous polypeptide and the LS-DNase amino acid sequence. It is expressed as a fusion with a heterologous polypeptide, such as a polypeptide. The term "operably linked" refers to the covalent joining of two or more sequences by enzymatic ligation or other means at a position relative to another sequence such that the normal function of the sequence can be performed. To do. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide when the polypeptide is expressed as a preprotein that participates in its secretion; the promoter or enhancer determines if it encodes If it has affected the transcription of the sequence, it is operably linked to the coding sequence; or the ribosome binding site is operably linked to the coding sequence if it is located so as to facilitate translation. ing. Generally, the term "operably linked" means that the DNA sequences being linked are contiguous, and in the case of a secretory leader, contiguous but still in the reading frame. Linking is accomplished by ligation at convenient restriction sites. And if the site is not present, a synthetic oligonucleotide adapter or linker is used in conjunction with standard recombinant DNA methods. Prokaryotes (eg, E. coli, strains of Bacillus, Pseudomonas, and other bacteria) are preferred host cells for the first cloning step of the present invention. They are particularly useful for the rapid production of large amounts of DNA, the production of single-stranded DNA templates used for site-directed mutagenesis, and the DNA sequencing of the mutants produced. Polypeptides produced in prokaryotes will typically not be glycosylated. In addition, LS-DNases are eukaryotic host cells, including eukaryotic bacteria (e.g., yeast) or cells from animals or other multicellular organisms (e.g., Chinese hamster ovary cells, and other mammalian cells); It will be expressed in live animals (eg, cows, goats, sheep). Insect cells can also be used. Cloning and expression methodologies are well known in the art. Examples of prokaryotic and eukaryotic host organisms suitable for use in the production of LS-DNase, as well as starter expression vectors, are described, for example, in Shak et al., PCT Patent Application N. o. There is one disclosed in WO90 / 07572. To obtain expression of the LS-DNase, the expression vector of the present invention is introduced into a host cell by transformation or transfection, so that the resulting recombinant host cell is suitable for inducing a promoter. Culture in a modified nutrient medium to select for recombinant cells or to amplify LS-DNase DNA. Culture conditions such as temperature, pH, etc. have been used previously for host cells and will be apparent to those of skill in the art. The terms "transformation" and "transfection" are used interchangeably to refer to a process for introducing DNA into cells. Following transformation or transfection, the DNA will be integrated into the host cell genome or will be present as an extrachromosomal element. If prokaryotes or cells containing substantial cell wall structures were used as hosts, the preferred method of transfection of cells with DNA is described by Cohen et al., Proc. Natl. Acad. Sci. 69: 2110-2114 (1972), or the Chung et al., Nuc. Acids. Res. 16: 3580 (1988). If yeast was used as the host, transfection was generally performed by Hinnen, Proc. Natl. Acad. Sci. U. S. A. , 75: 1929-1933 (1978). If mammalian cells were used as host cells, transfection was generally performed by Graham et al., Virology 52: 546 (1978), Gorman et al., DNA and Protein Eng. Tech. 2: 3-10 (1990). However, other well-known methods for introducing DNA into prokaryotic and eukaryotic cells such as intranuclear injection, electroporation, or protoplast fusion are also suitable for use with the present invention. Expression vectors that provide for the transient expression in mammalian cells of DNA encoding LS-DNase are particularly useful in the present invention. In general, transient expression is efficient in host cells where the host cell accumulates many copies of the expression vector, followed by high-level synthesis of the desired polypeptide encoded by the expression vector. Includes the use of replicable expression vectors. Transient expression systems, including suitable expression vectors and host cells, permit convenient positive identification of the polypeptide encoded by the cloned DNA, as well as those polypeptides having desirable biological or physiological properties. Allows for rapid screening of peptides. Wong et al., Science 228: 810-815 (1985); Lee et al., Proc. Natl. Acad. Sci. USA 82: 4360-4364 (1985); Yang et al., Cell 47: 3-10 (1986). Therefore, transient expression systems identify variants with useful properties such as increased half-life or decreased immunogenicity in vivo, increased DNA hydrolytic activity, or increased resistance to inhibition by actin In conjunction with the assay, it is conveniently used to express DNA encoding the amino acid sequence of a variant of LS-DNase. Inhibition of DNase activity by actin is readily measured using assays and methods well known in the art as described herein. LS-DNase is selected from the host cell that expresses it, in which case the variant is recovered from the culture medium in which the host cell is growing. In such a case, it is desirable to grow the cells in a serum-free culture medium because the absence of serum proteins and other serum components in the medium facilitates purification of the mutant. If not secreted, then LS-DNase is recovered from the lysate of the host cells. If the LS-DNase is expressed in a host cell other than that of human origin, the variant will be completely free of proteins of human origin. At any stage, it may be necessary to purify the LS-DNase from the recombinant cellular proteins in order to obtain a substantially homogeneous preparation of the LS-DNase. For therapeutic use, purified LS-DNase will preferably be greater than 99% pure (i.e., any other protein will contain less than 1% of the total protein in the purified composition). ). LS-DNase is described, for example, in PCT Patent Application No. It would be produced by a method involving homologous recombination and amplification, as described in WO 91/06667. Briefly, this method involves using homologous DNA to transform cells containing an endogenous gene encoding LS-DNase, which includes (1) an amplifiable gene (e.g., dihydrogenase). (2) a gene encoding folate reductase (DHFR), and (2) at least one flanking sequence having a length of at least about 150 base pairs, which is present in or near the gene encoding LS-DNase. Homologous to the nucleic acid sequence in the cell genome. Transformation is performed under conditions such that the homologous DNA is recombinantly integrated into the cell genome. The cells with the integrated homologous DNA are then subjected to conditions that select for amplification of the amplifiable gene, thereby simultaneously amplifying the LS-DNase gene. The resulting cells are then screened for production of the desired amount of LS-DNase. The flanking sequence present in the vicinity of the gene encoding LS-DNase can be easily identified, for example, by a genome walking method using the nucleic acid sequence of LS-DNase shown in FIG. 1 as a starting point. Spoerel et al., Meth. Enzymol. 152: 598-603 (1987). In general, purification of LS-DNase is accomplished by taking advantage of the different physical properties of LS-DNase as compared to the mixture with which it will be associated. For example, as a first step, the culture medium or host cell lysate is centrifuged to remove specific cell debris. The LS-DNase is then precipitated, e.g., by ammonium sulfate or ethanol precipitation, Kell filtration (molecular elution chromatography), ion exchange chromatography, hydrophobic chromatography, immunoaffinity chromatography (e.g., including anti-LS-DNase antibodies conjugated to Sepharose). Columns), tentacle cation exchange chromatography (Frenz et al., U.S. Pat.No. 5,279,823, filed Jan. 18, 1994), reverse phase HPLC, and / or gel electrophoresis. Purify from proteins and polypeptides. In some host cells, particularly bacterial host cells, LS-DNase is initially produced in an insoluble, aggregated form (referred to in the art as `` refractile bodies '' or `` inclusion bodies ''). It may be necessary to solubilize and reconstitute the LS-DNase under the assumption of purification. Methods for solubilizing and reconstituting recombinant protein reflective bodies are well known in the art (see Builder et al., US Pat. No. 4,511,502, filed Apr. 16, 1985). In another embodiment of the invention, LS- is used to confer desired properties (e.g., increased half-life or decreased in vivo immunogenicity, increased DNA hydrolytic activity, or increased resistance to inhibition by actin). The DNase is directly covalently modified, and the covalent modification can be made instead of or in addition to the amino acid substitution, insertion, and deletion mutations described above. Covalent modifications are introduced by reaction with the target amino acid of LS-DNase using an organic derivatizing reagent capable of reacting with the selected amino acid side chain or the N- or C-terminal side chain. Covalent coupling of glycosides to amino acid residues of the protein can be used to modify or increase the number or profile of carbohydrate substitutions. Covalent attachment of reagents such as polyethylene glycol (PEG) or human serum albumin to LS-DNase reduces the immunogenicity and / or toxicity of LS-DNase, as observed with other proteins And / or increase its half-life. Abuchowski et al., J. Biol. Chem. 252: 3582-3586 (1977); Pozansky et al., FE BS Letters 239: 18-22 (1988); Goodson et al., Biotechnolgy 8: 343-346 (1990); Katre, J. Im munol. 144: 209-213 (1990); Harris, Polyethylene Glycol Chehmistry (Plenum Press, 1992). In addition, modification of LS-DNase by these reagents at or near the amino acid residues that act on actin binding (i.e., within about 5 amino acid residues of the amino acid residues) is an increase in actin-induced inhibition. Will produce mutants with improved resistance. As another example, mutant or modified forms of LS-DNase include proteases (e.g., neutrophils) that may be present in sputum and other biological materials as compared to human LS-DNase. Amino acid sequence mutations or other covalent modifications that reduce the susceptibility of the mutant to degradation by globular elastase). An antibody against LS-DNase is optionally conjugated to an immunogenic polypeptide to immunize the animal with LS-DNase or a fragment thereof, and then recover the antibody from the serum of the immunized animal. Produced by Alternatively, monoclonal antibodies are prepared in a conventional manner from the cells of the immunized animal. The antibodies can also be made in the form of chimeric (eg, humanized) or single chain antibodies or Fab fragments using methods well known in the art. Preferably, the antibody will bind to LS-DNase, but will not substantially bind to (ie, cross-react with) other DNase proteins (such as human and bovine DNase I). The antibodies can be used in methods relating to LS-DNase localization and activity, for example, to detect LS-DNase and determine its level in tissue or clinical samples. Immobilized anti-LS-DNase antibodies are particularly useful in clinical samples for diagnostic purposes and in the purification of LS-DNase, in the case of detection of LS-DNase. Purified LS-DNase is used to reduce the viscoelastic properties of DNA-containing substances such as sputum, mucus, or other pulmonary secretions. LS-DNase has abnormal viscous or concentrated secretions and acute or chronic bronchi, including infectious pneumonia, bronchitis or tracheobronchitis, bronchiectasis, cystic fibrosis, asthma, tuberculosis, and fungal infection It is particularly useful for treating patients with lung disease who have a disease such as lung disease. For the treatment, a solution of LS-DNase or a homogeneously divided dry preparation is introduced into the patient's trachea (eg, bronchi) or lungs in a conventional manner, eg, by aerosol. LS-DNase is also useful in adjunctive treatment of ulcers, or for purulence, meningitis, ulcers, peritonitis, sinusitis, otitis, periodontitis, pericarditis, pancreatitis, cholelithitis, Cut off nearby infections in diseases such as endocarditis and septic arthritis, as well as various inflammations and infectious lesions such as skin and / or mucosal infected lesions, systemic in trauma, ulcer lesions and burns. It is also useful in the treatment of LS-DNase will improve the efficacy of the antibodies used in treating the infection (eg, gentamicin activity is significantly reduced by reversible binding to intact DNA). LS-DNase may also be used in patients with cystic fibrosis, chronic bronchitis, asthma, pneumonia, or other pulmonary disorders, or whose breathing is assisted by a ventilator or other mechanical device, or It is also useful to prevent the new formation and / or exacerbation of respiratory infections that can occur in other patients who may have a respiratory infection, such as post-operative patients. LS-DNase is also useful for the treatment of lupus erythematosus (SLE), a life-threatening autoimmune disease characterized by the production of different autoantibodies. DNA is the major antigenic component of the immune complex. In this case, the LS-DNase would be given systemically to the affected patient, for example by intravenous, subcutaneous, intraarterial or intramuscular administration. Finally, LS-DNase is useful in the treatment of other non-infectious diseases, where there is accumulation of cellular debris containing cellular DNA, such as renal nephritis and tubular interstitial kidney disease . LS-DNase can be formulated according to well-known methods of preparing therapeutically useful compositions. Typically, the LS-DNase is formulated with a physiologically acceptable excipient (or carrier) for therapeutic use. The excipient is used, for example, to provide a liquid or sustained release formulation of LS-DNase. A preferred therapeutic composition is a solution of LS-DNase in a buffered or unbuffered aqueous solution, especially 1. PH7 containing 0 mM calcium chloride. 0 is an isotonic salt solution such as 150 mM sodium chloride. These solutions are particularly suited for use in commercially available nebulizers, including jet nebulizers and ultrasonic nebulizers that are useful for direct administration into the trachea or lungs of affected patients. Another preferred therapeutic composition is a dry powder of LS-DNase, preferably prepared by spray-drying a solution of LS-DNase, essentially as described in PCT application WO 95/23613. Is done. In all cases, it is desirable that the therapeutic composition be sterile. Preferably, the therapeutic composition is discarded in a container made of plastic or other non-glass material. In a further embodiment, the therapeutic composition includes cells that vigorously produce LS-DNase. The cells are introduced directly into the patient's tissue or encapsulated in a porous membrane and then implanted into the patient (Aebischer et al., U.S. Pat.No. 4,892,538, assessed Jan. 9, 1990; Aebischer et al., US Pat. No. 5,283,187, filed Feb. 1, 1994) provides for the transport of LS-DNase to sites within the patient's body in need of increased concentrations of DNA hydrolysis in each case. For example, a patient's own cells can be transformed in vivo or ex vivo, respectively, with DNA encoding LS-DNase, and then used to produce LS-DNase directly in the patient. A therapeutically effective amount of LS-DNase will depend, for example, on the amount of DNA and actin in the substance to be treated, the therapeutic purpose, the route of administration, and the condition of the patient. Thus, a physician will need to measure the dosage and modify the route of administration required for optimal therapeutic effect. In view of reduced inhibition by actin and the resulting DNA hydrolytic activity in the presence of actin relative to human DNase I, the amount of LS-DNase required to achieve a therapeutic effect is Would be less than the amount of human DNase required to achieve the same effect in the same conditions. Generally, a therapeutically effective amount of LS-DNase is about 0. A dose of 1 μg to about 5 mg of the variant, administered in a pharmaceutical composition as described herein. LS-DNase may optionally be an antibiotic, a bronchodilator, an anti-inflammatory reagent, a mucus lysate (e.g., n-acetyl-cysteine), an actin binding protein or an actin auxiliary protein (e.g., gelsolin; Matsudaira et al., Cell 54: 139-140 (1988); Stossel et al., PCT Patent Application WO 94/22465, printed on October 13, 1994; protease inhibitors; or gene therapeutics (e.g., cystic fibrosis transmembrane conductance regulator (CFTR) gene); Riordan et al. , Science 245: 1066-1073 (189)) in combination with or administered with one or more pharmacological agents used to treat the above listed diseases. The present invention also provides a method for determining the presence of a nucleic acid molecule encoding LS-DNase in a test sample prepared from a cell, tissue, or biological fluid, comprising a nucleic acid coding sequence for LS-DNase. Contacting the test sample with the isolated DNA containing all or a portion of the test sample, and determining whether the isolated DNA hybridizes to a nucleic acid molecule in the test sample. DNA containing all or a portion of the nucleic acid coding sequence for LS-DNase is also substantially identical to the coding sequence for LS-DNase, such as a nucleic acid encoding a naturally occurring allelic variant of LS-DNase. It is also used in hybridization assays to identify and isolate nucleic acids having equivalent sequences. Also provided is a method of amplifying a nucleic acid molecule encoding LS-DNase present in a test sample, which includes the use of an oligonucleotide having a portion of the nucleic acid coding sequence for LS-DNase as a primer in a polymerase chain reaction. It is. The following examples are provided by way of illustration only and are not intended to limit the invention in any way. All patent and literature references cited herein are expressly incorporated. Example 1 Cloning of LS-DNase cDNA A full-length cDNA encoding LS-DNase was purified from a human liver cDNA library (λ-UniZAP XR, Stratagene, La Jolla , In CA), each probe was identified as γ-specific for T4 polynucleotide kinase and highly specific radioactivity. ³⁵ End-labeled with P-adenosine triphosphate: The first three of the oligonucleotide probes listed above (SEQ.ID.NOS: 5-7) have a portion of the EST sequence with registration codes T68985, T69063, HSAAACIFW, T73558, T61400, T7 3653 and T61368 in the GENbank database. Is included. The other two oligonucleotides listed above (SEQ. ID. NOS: 9-10) include portions of the EST sequence with accession codes R78020 and H42990 in the Genbank database. Hybridization of the probe to the cDNA library was digested at 42 ° C. under low stringency conditions (20% vol / vol formamide, 5 × SSPE, 5 × Denhardt's solution, 0.1% sodium dodecyl sulfate (SDS), and 100 μg / ml). Salmon sperm DNA). Post-hybridization washes were performed at 42 ° C. in 2 × SSC, 0.1% SDS. 1 × SSPE is 150 mM NaCl, 10 mM sodium phosphate, 1 mM EDTA, pH 7.4. 1x Denhardt's solution is 0.02% Ficoll, 0.02% bovine serum albumin, and 0.02% polyvinylpyrrolidone. 1 × SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0. Hybridization positive clones were found only with the first three oligonucleotides listed above (SEQ. ID. NOS: 5-7). These clones were converted into phagemid base sequences according to standard methods (Stratagene, La Jolla, California, USA) and sequenced. The largest inserted nucleic acid sequence found in the hybridization positive clones was 1079 base pairs long, including a 915 base pair open reading frame encoding a protein predicted to be 305 amino acids long. The nucleic acid sequence of the 1079 base pair insert (SEQ.ID.NO:1) and the amino acid sequence of the predicted protein (SEQ.ID.NO:2) are shown in FIG. The predicted protein contains a signal sequence 20 amino acids in length. Cleavage of the signal sequence releases the mature protein (LS-DNase), which has a predicted molecular weight of 33,400 daltons and a predicted pi of 9.7. The amino acid sequence of LS-DNase is 46% identical to that of human DNase I (FIG. 2). LS-DNase contains five cysteine residues, two of which (Cys-174 and Cys-211) correspond to a pair of cysteine residues in human DNase I that are disulfide-bonded, which This suggests that human DNase I has the same quaternary structure. Amino acid residues known to be important for the DNA hydrolytic activity of human DNase I are also conserved in LS-DNase, including the active site histidine residue His-135. And His-254. Conversely, several amino acid residues known to be included in the actin binding site of human DNase I are conserved in LS-DNase. In particular, Val-67 and Ala-114 of human DNase I have been replaced by Ile-69 and Phe-115, respectively, at the homologous positions in LS-DNase. Analog replacement of Val-67 by Ile has occurred in rat DNase I, which has an approximately 1000-fold lower affinity for actin compared to human DNase I. Example 2 Expression of LS-DNase cDNA cDNA encoding LS-DNase was prepared using the mammalian vector pRK5 (Gorman et al., DNA and Protein Engineering Techniques 2: 1 (1990); Europe printed on March 15, 1989. Subcloned in patent application EP 307,247). The resulting recombinant vector is called pRK5 / LS-DNase. Human embryonic kidney 293 cells (American Type Culture Collection, CRL 1573) were grown in Dulbecco's modified Eagle's medium containing serum (DMEM) to 70% coverage (70% confluency) and then pRK5 / LS-DNase, or Transfected with pRK5 alone as a control. Twenty-four hours after transfection, the cells were washed with phosphate buffered saline and transferred to a serum-free medium containing insulin. After 72-96 hours, the conditioned media was collected from the cell culture and concentrated approximately 10-fold. The proteins expressed in the cell culture were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Cells transfected with pRK-5 / LS-DNase were found to produce approximately 32,000-34,000 daltons of a unique, readily lysable protein, which was transfected with pRK5 alone It was not produced in cells. The molecular weight of this protein is in good agreement with that expected for LS-DNase. Example 3 Biological activity of LS-DNase The concentrated cell culture supernatant prepared as described above was subjected to a hyperchromicity assay (Kunitz, J. Gen. Physiol. 33: 349- 36 2 (1950); Kunitz, J. Gen. Physiol. 33: 363-377 (1950)) and methyl green assay (Kurnick, Arch.Biochem. 29: 41-53 (1950); Sunicropi et al., Anal.Biochem. 222: 351-358 (1994)). In both assays, DNase activity was detected in supernatants from cells transfected with pRK5 / LS-DNase but not in cells from cells transfected with pRK5 alone. Was. Example 4 Resistance to Actin Inhibition To determine whether actin inhibits the DNA hydrolytic activity of LS-DNase, a plasmid nicking assay was used. This assay measures the conversion of supercoiled double-stranded pBR322 plasmid DNA to nicked, linearized, and degraded forms. Specifically, various DNases were added in 25 mM HEPES buffer, 1 mM MgCl _Two , 1mMCaCl _Two Was added to 20 μl of a solution containing 25 μg / ml supercoiled double-stranded pBR322 DNA in 100 μg of bovine serum albumin, and the sample was incubated at 21 ° C. for 10 minutes. To measure inhibition by actin, the DNase sample was pre-incubated with actin at 21 ° C. for 15 minutes before adding a solution of pBR322 DNA. The reaction was stopped by adding 10 mM final concentration of EDTA while simultaneously adding xylene cyanol, bromophenol and glycerol. The integrity of ± BR322 DNA was analyzed by electrophoresis of the reaction mixture on a 0.8% weight / vol agarose gel. After electrophoresis, the gel was stained with a solution of ethidium bromide, and the DNA in the gel was visualized by ultraviolet light. As expected, human DNase I converted the start plasmid DNA into a degraded form, and the DNA hydrolyzing activity of human DNase I was inhibited by actin added in a concentration-dependent manner. Although LS-DNase converted the start plasmid DNA into a nicked, linearized, and degraded form, the DNA hydrolysis activity of LS-DNase was not inhibited by the concentration of actin that sufficiently inhibited human DNase I. Was. Example 5 Expression Pattern of LS-DNase in Human Tissues Northern blots of various human tissues were performed using a radiolabeled probe containing a portion of the cDNA coding sequence of the cloned LS-DNase. Expression of LS-DNase messenger RNA (mRNA) was found to be highest in liver and spleen. LS-DNase was expressed poorly or not at all in the other tissues examined. LS-DNase mRNA was not detected at all in pancreatic tissue. Northern blots of various human tissues were also performed with a radiolabeled probe containing a portion of the nucleic acid coding sequence for human DNaseI. In contrast to LS-DNase mRNA, human DNase I mRNA was found to be extremely expressed in pancreatic tissue. Example 6 Cloning of LS-DNase Variants A 649 base pair EcoRI-PStI fragment of the coding sequence of the cloned LS-DNase cDNA was prepared using a murine liver cDNA library (λ-gt10, Clontech, Palo Alto, California, USA). ) Was used for screening. From about 2,000,000 clones screened, more than 60 hybridization positive clones were identified. Partial sequencing of 6 random positive clones showed that they all came from the same gene. One inserted nucleic acid sequence of the positive clone was completely sequenced. The insert is 1124 base pairs in length, contains the 930 base pair-open reading frame encoding the predicted protein, and is 310 amino acids long and is called murine LS-DNase. The nucleic acid sequence of the 1124 base pair insert (SEQ. ID. NO: 11) is shown in FIG. The open reading frame starts with the ATG codon at nucleic acid 173 and continues to the stop codon at nucleic acid 1103. The first 75 nucleic acids of the open reading frame (the first 25 amino acid residues of the predicted protein) encode a putative signal sequence. Thus, the predicted murine mature LS-DNase protein is 285 amino acids long, has a molecular weight of 33,100 daltons and a predicted pI of 9.4. The amino acid sequence of murine mature LS-DNase is 84% identical to the amino acid sequence shown in FIG. 1 for human mature LS-DNase. Northern blots of various mouse tissues were performed with a radiolabeled probe containing a portion of the nucleic acid coding sequence for murine LS-DNase. Expression of murine LS-DNase messenger RNA (mRNA) was found to be highest in liver and spleen. LS-DNase mRNA was expressed slightly or not at all in the other tissues examined.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 48/00 C07K 16/40 C07K 16/40 C12N 9/22 C12N 5/10 C12P 21/08 9/22 C12N 5/00 B C12P 21/08 A61K 37/54 (81) Designated country EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL , PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, LS, MW, SD, SZ, UG), UA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, H, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, HU, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT , LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TR, TT, UA, UG, UZ, VN, YU

Claims (1)

[Claims] 1． Nucleotide encoding the amino acid sequence shown in FIG. 1 for mature LS-DNase An isolated nucleic acid molecule comprising a tide sequence. 2． A vector recognized by a host cell transformed with a vector As shown in Figure 1 for mature LS-DNase, operably linked to An expression vector comprising a nucleotide sequence encoding an amino acid sequence. 3． At least 95% identical to the amino acid sequence shown in Figure 1 for the mature LS-DNase Isolated nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence having sex . Four. Substitution of one amino acid by another amino acid only at a single position in the sequence of Figure 1 Therefore, amino acids that differ from the amino acid sequence shown in FIG. 1 for mature LS-DNase An isolated nucleic acid molecule comprising a nucleotide sequence encoding an acid sequence. Five. Substitution of one amino acid by another amino acid only at position 2 in the sequence of FIG. Thus, amino acids that differ from the amino acid sequence shown in Figure 1 for the mature LS-DNase An isolated nucleic acid molecule comprising a nucleotide sequence that encodes the sequence. 6． Nucleotide encoding the amino acid sequence shown in FIG. 1 for mature LS-DNase A host cell transformed with an expression vector containing a peptide sequence. 7． Nucleotide encoding the amino acid sequence shown in FIG. 1 for mature LS-DNase A method of using a host cell transformed with an expression vector containing a peptide sequence, Culturing the host cell under conditions such that the expression vector is replicated Method. 8． A polypeptide comprising the amino acid sequence shown in FIG. 1 for mature LS-DNase Ko Transforming a host cell with the nucleic acid molecule to be loaded, wherein the polypeptide is Culturing the host cells under conditions such that they are produced in the host cells Process. 9． (A) cells containing an endogenous LS-DNase gene, an amplifiable gene and endogenous AL-1 At least about 150 base pairs homologous to the DNA sequence within or adjacent to the gene. Transformation using homologous DNA containing the flanking sequence of This allows the homologous DNA to integrate into the cell genome by recombination: (b) by culturing the cells under conditions selected for amplification of the amplifiable gene, Thereby the LS-DNase gene is also amplified; and (c) recovering LS-DNase from the cells LS-DNase production method including Ten. An isolated polymorph comprising the amino acid sequence shown in FIG. 1 for the mature LS-DNase peptide. 11． Has 95% identity to the amino acid sequence shown in Figure 1 for mature LS-DNase An isolated polypeptide comprising an amino acid sequence, wherein said polypeptide is DNA hydrolyzed An active polypeptide. 12． Substitution of one amino acid by another amino acid only at a single position in the sequence of FIG. Depending on the amino acid sequence that differs from the amino acid sequence shown in Figure 1 for the mature LS-DNase. An isolated polypeptide comprising a nucleic acid sequence. 13. Groups in polypeptides where the amino acid substitution is not present in human LS-DNase 13. The polypeptide of claim 12, which creates a lycosylation site. 14. Amino acid sequence shown in Figure 1 for mature LS-DNase and pharmaceutically acceptable A pharmaceutical composition comprising a polypeptide comprising an excipient to be prepared. 15． 15. The composition according to claim 14, which is sterile. 16． An antibody capable of binding to the amino acid sequence shown in FIG. 1 against mature LS-DNase. 17． 17. The antibody according to claim 16, which is a monoclonal antibody. 18． A pulmonary disease or disorder comprising administering to the patient a therapeutically effective amount of LS-DNase; How to treat the patient with harm. 19. 19. The method according to claim 18, wherein said disease or disorder is cystic fibrosis. 20. Systemic lupus erythematosus comprising administering to a patient a therapeutically effective amount of LS-DNase Treatment of said patient with acne.